ABSTRACT Accumulating evidence suggests that spinal astrocytes play an important role in the genesis of persistent pain, by increasing the activity of spinal cord nociceptive neurons, i.e., central sensitization. However, direct evidence of whether activation of astrocytes is sufficient to induce chronic pain symptoms is lacking. We investigated whether and how spinal injection of activated astrocytes could produce mechanical allodynia, a cardinal feature of chronic pain, in naïve mice. Spinal (intrathecal) injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by tumor necrosis factor-alpha (TNF-α), induced a substantial decrease in paw withdrawal thresholds, indicating the development of mechanical allodynia. This allodynia was prevented when the astrocyte cultures were pretreated with a peptide inhibitor of c-Jun N-terminal kinase (JNK), D-JNKI-1. Of note a short exposure of astrocytes to TNF-α for 15 min dramatically increased the expression and release of the chemokine monocyte chemoattractant protein-1 (MCP-1), even 3 h after TNF-α withdrawal, in a JNK-dependent manner. In parallel, intrathecal administration of TNF-α induced MCP-1 expression in spinal cord astrocytes. In particular, mechanical allodynia induced by TNF-α-activated astrocytes was reversed by a MCP-1 neutralizing antibody. Finally, pretreatment of astrocytes with MCP-1 siRNA attenuated astrocytes-induced mechanical allodynia. Taken together, our results suggest that activated astrocytes are sufficient to produce persistent pain symptom in naïve mice by releasing MCP-1.

injured regions and even contralateral side of body parts (Baron, 2009; Campbell et al.,1988). It is generally believed that hyperactivity in the spinal cord pain circuit, i.e. centralsensitization, contributes to the generation of mechanical allodynia and spread of pain (Ji etal., 2003; Woolf and Salter, 2000).Recent progress in pain research has pointed to an important role of glial cells in thegeneration of chronic pain. Many lines of evidence indicate that glial cells such as microgliaand astrocytes in the spinal cord become reactive in chronic pain conditions and contributeto the development and maintenance of chronic pain by inducing central sensitization(DeLeo and Yezierski, 2001; Garrison et al., 1994; Ji and Strichartz, 2004; McMahon andMalcangio, 2009; Milligan and Watkins, 2009; Ren and Dubner, 2008; Scholz and Woolf,2007). Accumulating evidence demonstrates that activation of microglia in the spinal cordinduces neuropathic pain by producing proinflammatory cytokines and the brain-derivedneurotrophic factor (BDNF) (Coull et al., 2005; Inoue and Tsuda, 2009; Suter et al., 2007;Tsuda et al., 2005; Tsuda et al., 2003). The role of astrocytes in chronic pain and theunderlying mechanisms have also been investigated (Ji et al., 2006). Astrocytes areorganized in gap junction-coupled networks. They not only transmit Ca2+ signaling in theform of oscillations or waves through the networks (Haydon, 2000), but also form a“tripartite” synapse with pre- and post-synaptic membranes and through which modulatesynaptic strength (Haydon and Carmignoto, 2006; Jourdain et al., 2007). After injuries,reactive astrocytes express the c-Jun-N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK), and produce the proinflammatory cytokineinterleukin-1beta (IL-1β) and chemokine MCP-1, enhancing and maintaining centralsensitization and chronic pain states (Gao et al., 2009; Guo et al., 2007; Kawasaki et al.,2008a; Kawasaki et al., 2008b; Ren and Dubner, 2008; Zhuang et al., 2006).Spinal injection of ATP-activated microglia has been shown to produce mechanicalallodynia via releasing BDNF (Coull et al., 2005; Tsuda et al., 2003). It remains unclearwhether and how activated astrocytes are sufficient to induce this persistent pain symptom.Our recent study showed that TNF-α induced a dramatic increase of MCP-1 in astrocytes viathe activation of JNK (Gao et al., 2009). In this study we further examined whether TNF-α-activated astrocytes would induce mechanical allodynia by releasing MCP-1.Materials and MethodsAnimalsCD1 mice, obtained from Charles River Laboratories, were used for most experiments.Adult CD1 mice (male, 25–32 g) were used for behavioral studies. Neonatal CD1 mice (P2)were used to prepare primary cultures of astrocytes. TNF receptor (R1/R2) double knockoutmice (TNFR1/R2−/−, male, 25–32 g), obtained from Jackson Laboratories, and C57BL/6wild-type control mice were also used in some experiment. All animal procedures performedin this study were approved by the Animal Care Committee of Harvard Medical School.ReagentsTNF-α was purchased from R&D. MCP-1 neutralizing antibody and control serum werepurchased from Millipore and Invitrogen, respectively. The JNK inhibitor D-JNKI-1 waskindly provided by Dr. Christopher Bonny, University of Lausanne, Switzerland (Borsello etal., 2003). MCP-1 siRNA was purchased from Santa Cruz. Non-targeting siRNA wassynthesized by Dharmacon Research Incorporation as a control siRNA. SiRNA wasdissolved in RNase-free water at 1 µg/µl as stock solution and mixed with the transfectionreagent polyethyleneimine (PEI, Fermentas Inc) and normal saline before use. Specifically,Gao et al.Page 2Glia. Author manuscript; available in PMC 2011 November 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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1 µg siRNA was dissolved in 3.3 µl PEI and 66 µl normal saline (Tan et al., 2009; Tan et al.,2005).Primary culture of astrocytesTo get high quality and large quantity of astrocytes, we prepared most astrocyte culturesfrom cerebral cortexes of neonatal mice (P2). We also prepared some astrocyte culturesfrom spinal cords of neonatal mice. After dissection, we transferred the cerebralhemispheres or spinal cord segments to ice-cold Hank’s buffer and carefully removed themeninges. Tissues were then minced into ~1 mm pieces, triturated, filtered through a 100µm nylon screen, and collected by centrifugation at ~3000g for 5 min. The cell pellets werebroken with a pipette and resuspended in a medium containing 15% fetal bovine serum(FBS) in low glucose Dulbecco's Modified Eagle's Medium (DMEM). After trituration, thecells were filtered through a 10 µm screen and then plated onto 6-well plates at a density of2.5 × 105 cells/cm2, and cultured for 10–12 days. The medium was replaced twice a week,first with 15% FBS, then with 10% FBS. Once the cells were grown to about 95%confluence, 0.15 mM dibutyryl cAMP (Sigma) was added to induce differentiation. Threedays later, the cells were used for experiments.Intrathecal administrationBefore injection, astrocytes were washed with 0.01 M phosphate buffer saline (PBS) for 3times and centrifuged for 5 min at 3000 g. Astrocytes were then resuspended in PBS. Forintrathecal injection, spinal cord puncture was made with a 30G needle between the L5 andL6 level to deliver the cells or reagents (10 µl) to the cerebral spinal fluid (Hylden andWilcox, 1980).ELISAMouse MCP-1 ELISA kit was purchased from R&D. Culture medium and cells werecollected separately after treatment. Astrocytes were homogenized in a lysis buffercontaining protease and phosphatase inhibitors (Zhuang et al., 2006). Protein concentrationswere determined by BCA Protein Assay (Pierce). For each reaction in a 96-well plate, 100µg of proteins or 50 µl of culture medium were used, and ELISA was performed accordingto manufacturer’s protocol. The standard curve was included in each experiment.ImmunohistochemistryAnimals were deeply anesthetized with isoflurane and perfused through the ascending aortawith PBS followed by 4% paraformaldehyde with 1.5% picric acid in 0.16 M PB. After theperfusion, the L4–L5 spinal cord segments were removed and postfixed in the same fixativeovernight. Spinal cord sections (30 µm, free-floating) were cut in a cryostat and processedfor immunofluorescence as we described previously (Jin et al., 2003; Zhuang et al., 2006).For Iba1 staining, spinal cord sections were first blocked with 2% goat serum for 1 h atroom temperature, then incubated overnight at 4°C with rabbit anti-Iba1 primary antibody(1:5000, Wako), followed by incubating with Cy3-conjugated secondary antibody (1:400,Jackson ImmunoResearch) for 1 h at room temperature. For double staining of MCP-1 andGFAP, the spinal cord sections were blocked with 2% goat serum and then incubated with amixture of primary antibodies against MCP-1 (rabbit, 1:500, Millipore) and GFAP (mouse,1:5000, Millipore), followed by a mixture of corresponding secondary antibodies conjugatedwith either Cy3 or FITC (1:400, Jackson ImmunoResearch) (Gao et al., 2009). The stainedspinal cord sections were examined with a Nikon fluorescence microscope, and images werecaptured with a CCD Spot camera.Gao et al.Page 3Glia. Author manuscript; available in PMC 2011 November 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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Behavioral analysisAnimals were habituated to the testing environment daily for at least two days beforebaseline testing. The room temperature and humidity remained stable for all experiments.For testing mechanical sensitivity, animals were put in boxes on an elevated metal meshfloor and allowed 30 min for habituation before examination. The plantar surface of eachhindpaw was stimulated with a series of von Frey hairs with logarithmically incrementingstiffness (0.02–2.56 grams, Stoelting), presented perpendicular to the plantar surface (1–2seconds for each hair). The 50% paw withdrawal threshold was determined using Dixon’sup-down method (Chaplan et al., 1994).Quantification and statisticsFive nonadjacent spinal cord sections were randomly selected from a spinal cord segment(L4–L5) and 3 mice were included for each group. The intensity of Iba1 staining in thesuperficial dorsal horn (laminae I–III) was measured with a computer-assisted imaginganalysis system (Image J, NIH). The number of MCP-1 positive cells in the spinal corddorsal horn (laminae I–III) was also counted under microscope. All the data were expressedas mean ± s.e.m. Differences between groups were compared by one-way ANOVA withrepeated measurement and student t-test. The criterion for statistical significance wasP<0.05.ResultsIntrathecal administration of TNF-α-stimulated astrocytes induces mechanical allodyniaTo determine whether activated astrocytes are sufficient to induce pain sensitization, weprepared primary astrocyte cultures from cerebral cortexes of neonatal mice (P2) anddifferentiated them with 0.15 mM dibutyryl cAMP to mimic mature astrocytes. Weactivated astrocytes with TNF-α to release MCP-1 (Gao et al., 2009). After a briefincubation with TNF-α (10 ng/ml, 15 min), we washed astrocytes 3 times with PBS toremove TNF-α and collected the astrocytes for intrathecal injection in naïve mice (Fig.1A).We found a dramatic reduction in paw withdrawal threshold (PWT) after inthathecalinjection of TNF-α-stimulated astrocytes, indicating the development of mechanicalallodynia (P<0.05, one-way ANOVA, Fig.1B). This allodynia developed at 3 h and lastedfor more than 48 h (Fig.1B). Notably, intrathecal injection of non-stimulated controlastrocytes also significantly reduced PWT (P<0.05, one-way ANOVA). However, the PWTfollowing injection of activated astrocytes was much lower than that following injection ofcontrol astrocytes (P<0.05, t-test, Fig. 1B).Because JNK is known to be activated by TNF-α and nerve injury in astrocytes andcontributes to the development of mechanical allodynia (Gao et al., 2009; Zhuang et al.,2006), we examined whether mechanical allodynia elicited by TNF-α-stimulated astrocyteswould require JNK. Pretreatment of cultured astrocytes with the peptide inhibitor of JNK,D-JNKI-1 (20 µM) starting 30 min before TNF-α stimulation, significantly reducedmechanical allodynia evoked by the activated astrocytes (P<0.05, t-test, Fig. 1B).Collectively, these results suggest that TNF-α-activated astrocytes are sufficient to inducemechanical allodynia in naïve mice, in a JNK-dependent manner.TNF-α induces MCP-1 expression and release in astrocytes via activation of JNKTo explore possible mechanisms underlying the astrocytes-induced tactile allodynia, weexamined MCP-1 expression and release in cultures using an experimental protocol tomimic in vivo conditions, as shown in Fig.2A. Brief incubation of astrocytes with TNF-α for15 min increased MCP-1 expression. Interestingly, even after we removed TNF-α by 3 timesPBS wash, MCP-1 expression continued to increase 3 h after initial TNF-α stimulation (15Gao et al.Page 4Glia. Author manuscript; available in PMC 2011 November 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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min only). This increase was partially prevented when the astrocytes were pretreated withD-JNKI-1 (20 µM, P<0.05, t-test, Fig.2B). The brief application of TNF-α (15 min) alsoincreased MCP-1 release in culture medium. Strikingly, 3 h after removal of TNF-α andreplaced it with fresh medium, MCP-1 release further increased 10 times (P<0.05, t-test),and this increase in release was also reduced by D-JNKI-1 pretreatment (P<0.05, t-test, Fig.2A, 2B).To examine if astrocytes from the spinal cords have similar response to TNF-α as astrocytesfrom the cortexes, we prepared astrocyte cultures from the spinal cords and incubated thecultures with TNF-α for 15 min. ELISA analysis showed that a brief exposure of TNF-α alsoinduced a substantial increase in MCP-1 expression and release in spinal cord astrocytes(Supporting information Fig.1). Thus, cortical astrocytes and spinal cord astrocytes showsimilar responses to TNF-α by inducing robust MCP-1 expression and release.TNF-α induces MCP-1 expression in spinal cord astrocytesTo further determine whether astrocytes in spinal cord in vivo show similar response toTNF-α as cultured astrocytes, we examined whether intrathecal TNF-α can induce MCP-1expression in the intact spinal cord. Intrathecal TNF-α at a dose (20 ng) that is known toelicit mechanical allodynia (Gao et al., 2009) markedly increased MCP-1 expression in thespinal cord 3 h after the injection (Fig. 3A,B). The number of MCP-1-positive cells in thesuperficial dorsal horn (laminae I–III) increased from 10.4 ± 0.2 cells per section in thePBS-treated group to 33.5 ± 2.4 cells per section in the TNF-α-treated group (P<0.05, t-test,n=3 mice). Double staining revealed that MCP-1 was co-localized with the astrocyte markerGFAP (Fig.3E, F). These data strongly suggest that TNF-α induces MCP-1 expression notonly in cultured astrocytes but also in spinal cord astrocytes in vivo, in support of ourprevious result that nerve injury induces MCP-1 in spinal cord astrocytes (Gao et al., 2009).MCP-1 neutralizing antibody reverses mechanical allodynia induced by activatedastrocytes but not by control astrocytesTo test the hypothesis that TNF-α-activated astrocytes release MCP-1 to generate tactileallodynia in naïve animals, we intrathecally injected a MCP-1 neutralizing antibody at 3 hafter intrathecal injection of TNF-α-treated astrocytes. At a dose (5 µg) that is effective inreducing SNL-induced neuropathic pain (Gao et al., 2009) the neutralizing antibodyreversed the mechanical allodynia induced by activated astrocytes (P<0.05, one-wayANOVA, Fig. 4A). This reversal began at 30 min, maintained at 3 h, but diminished at 24 hfollowing the antibody injection (Fig. 4A). In contrast, intrathecal injection of the controlserum had no effect on mechanical allodynia (P>0.05, one-way ANOVA, Fig. 4A).Since intrathecal injection of control astrocytes also decreased paw withdrawal threshold innaïve animals (Fig.1B), we further checked if this allodynia might also depend on MCP-1.Intrathecal injection of the MCP-1 neutralizing antibody (5 µg) did not change the PWT at 3h after injection of control astrocytes (P>0.05, one-way ANOVA, Fig.4B). Thus, mechanicalallodynia elicited by control astrocytes does not require MCP-1.Intrathecal injection of TNF-α is known to elicit mechanical allodynia (Gao et al., 2009). Toexclude the possibility that the mechanical allodynia induced by TNF-α-stimulatedastrocytes is caused by the residue TNF-α in the culture, we injected the TNF-α-stimulatedastrocytes into double knockout mice lacking type I and II TNF receptors (TNFR1/R2 −/−)using C57BL/6 wild-type mice as control. Notably, intrathecal injection of TNF-α-stimulated astrocytes was still able to elicit mechanical allodynia in knockout mice (P>0.05,t-test, supporting information, Fig.2). This finding suggests that mechanical allodyniaGao et al. Page 5Glia. Author manuscript; available in PMC 2011 November 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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elicited by activated astrocytes is caused neither by residue TNF-α in the culture mediumnor by TNF-α released from astrocytes.Astrocytes-induced mechanical allodynia is reduced by pretreatment of astrocytes withMCP-1 siRNATo further confirm the role of astrocytic MCP-1 release in generating mechanical allodynia,we treated astrocyte cultures with a specific small interfering RNA (siRNA) against MCP-1.Astrocytes were incubated with MCP-1 siRNA for 18 h followed by TNF-α stimulation for15 min, and the medium and cell lysates were collected 3 h later. MCP-1 siRNA treatmentinhibited TNF-α-induced MCP-1 expression and release in a dose-dependent manner (Fig.5A, B). At the concentration of 0.1 and 1 µg/ml, MCP-1 siRNA inhibited MCP-1 expressionby 22% and 68% and MCP-1 release by 32% and 73%, respectively, compared to non-treated astrocytes. Notably, the non-targeting siRNA at the high concentration (1µg/ml) alsodecreased MCP-1 expression and release by 28%. This effect of non-targeting siRNA maybe associated with interferon-mediated off-target effects (Sledz et al., 2003). However,MCP-1 siRNA at the high concentration (1 µg/ml) produced more robust inhibition ofMCP-1 expression and release (P<0.01, t-test, Fig. 5A, B).After we confirmed an effective knockdown of MCP-1 expression and release by MCP-1siRNA in cultured astrocytes, we next examined the effect of this knockdown on painbehavior. Following incubation with MCP-1 siRNA or non-targeting control siRNA (1 µg/ml, 18 h), astrocytes were stimulated with TNF-α for 15 min, washed with PBS and thencollected for intrathecal injection in naïve mice. Mechanical allodynia was reduced after theinjection of MCP-1 siRNA-treated astrocytes compared with that after the injection ofcontrol siRNA-treated astrocytes (P<0.05 or P<0.01, t-test, Fig. 5C).Expression of the microglial marker Iba1 in the spinal cord does not change afterintrathecal injection of activated astrocytesTo determine possible mechanisms by which astrocyte-released MCP-1 producesmechanical allodynia, we examine microglial reaction in the spinal cord, because MCP-1has been shown to promote neuropathic pain by inducing microglial responses in the spinalcord (Thacker et al., 2009; Zhang et al., 2007). We collected spinal cord segments at 48 hafter intrathecal injection of TNF-α-stimulated astrocytes or control astrocytes and examinedthe expression of the microglial marker, Iba1, which is associated with microglialproliferation and migration (Zhang et al., 2007). To our surprise, we did not observe anydifference in the intensity of Iba1 immunofluorescence between these two groups (P>0.05, t-test). Neither did we find any differences in the morphology of Iba1-stained microglia (Fig.6A–D). Therefore, there is no evidence of microgliosis in the spinal cord followingintrathecal injection of activated astrocytes.DiscussionAstrocytes are required for producing chronic pain symptomsAstrocytes become reactive in chronic pain conditions after nerve injury and tissue injury/inflammation (DeLeo et al., 2004; Garrison et al., 1994; Garrison et al., 1991; Raghavendraet al., 2004; Zhuang et al., 2006). Several lines of evidence suggest that astrocytes arerequired for the generation of persistent pain. First, intrathecal injection of astroglial toxinfluorocitrate (Milligan et al., 2003) and L-alpha-aminoadipate (Zhuang et al., 2006) inhibitsnerve injury- or nerve inflammation-induced mechanical allodynia. Second, astrocytes formastroglial networks by gap junctions (Giaume and McCarthy, 1996), and intrathecal gapjunction blocker carbenoxolone suppresses mechanical allodynia in the contralateral pawafter nerve inflammation (Spataro et al., 2004). Third, intrathecal administration of theGao et al. Page 6Glia. Author manuscript; available in PMC 2011 November 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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[Show abstract][Hide abstract]ABSTRACT: The proinflammatory cytokines TNF- and IL-1 have been strongly implicated in the pathogenesis of neuropathic pain, but the intracellular signaling of these cytokines in glial cells are not fully understood. Tumor necrosis factor receptor associated factor 6 (TRAF6) plays a key role in signal transduction in the TNF receptor superfamily and the interleukin-1 receptor superfamily. In this study, we investigated the role of TRAF6 in neuropathic pain in mice following spinal nerve ligation (SNL). SNL induced persistent TRAF6 upregulation in the spinal cord. Interestingly, TRAF6 was mainly colocalized with the astrocytic marker GFAP on SNL day 10 and partially expressed in microglia on SNL day 3. In cultured astrocytes, TRAF6 was up-regulated after exposure to TNF-α or IL-1β. TNF-α or IL-1β also increased CCL2 expression, which was suppressed by both siRNA and shRNA targeting TRAF6. TRAF6 siRNA treatment also inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) in astrocytes induced by TNF-α or IL-1β. JNK inhibitor D-NKI-1 dose-dependently decreased IL-1-induced CCL2 expression. Moreover, spinal injection of TRAF6 siRNA decreased intrathecal TNF-- or IL-1-induced allodynia and hyperalgesia. Spinal TRAF6 inhibition via TRAF6 siRNA, shRNA lentivirus, or antisense oligodeoxynucleotides partially reversed SNL-induced neuropathic pain and spinal CCL2 expression. Finally, intrathecal injection of TNF-α-activated astrocytes induced mechanical allodynia, which was attenuated by pretreatment of astrocytes with TRAF6 siRNA. Taken together, the results suggest that TRAF6, upregulated in spinal cord astrocytes in the late phase after nerve injury, maintains neuropathic pain by integrating TNF- and IL-1 signaling and activating the JNK/CCL2 pathway in astrocytes.

"Numerous studies have indicated that astrocytes make major contributions to pain-related behavior following peripheral nerve injury and inflammation, and this extensive body of work is covered comprehensively by several recent reviews (Ellis and Bennett, 2013; Ji et al., 2013; Mika et al., 2013). Importantly, spinal injection of astrocytes that had been activated by TNFα was shown to be sufficient to produce mechanical hypersensitivity in uninjured animals (Gao et al., 2010). Far fewer studies have been made of astroglial contributions to neuropathic pain caused by SCI, but the findings thus far show interesting similarities to what has been described in peripheral neuropathic pain models. "

[Show abstract][Hide abstract]ABSTRACT: Neuropathic pain after spinal cord injury (SCI) is common, often intractable, and can be severely debilitating. A number of mechanisms have been proposed for this pain, which are discussed briefly, along with methods for revealing SCI pain in animal models, such as the recently applied conditioned place preference test. During the last decade, studies of animal models have shown that both central neuroinflammation and behavioral hypersensitivity (indirect reflex measures of pain) persist chronically after SCI. Interventions that reduce neuroinflammation have been found to ameliorate pain-related behavior, such as treatment with agents that inhibit the activation states of microglia and/or astroglia (including IL-10, minocycline, etanercept, propentofylline, ibudilast, licofelone, SP600125, carbenoxolone). Reversal of pain-related behavior has also been shown with disruption by an inhibitor (CR8) and/or genetic deletion of cell cycle-related proteins, deletion of a truncated receptor (trkB.T1) for brain-derived neurotrophic factor (BDNF), or reduction by antisense knockdown or an inhibitor (AMG9810) of the activity of channels (TRPV1 or Nav1.8) important for electrical activity in primary nociceptors. Nociceptor activity is known to drive central neuroinflammation in peripheral injury models, and nociceptors appear to be an integral component of host defense. Thus, emerging results suggest that spinal and systemic effects of SCI can activate nociceptor-mediated host defense responses that interact via neuroinflammatory signaling with complex central consequences of SCI to drive chronic pain. This broader view of SCI-induced neuroinflammation suggests new targets, and additional complications, for efforts to develop effective treatments for neuropathic SCI pain.

"Our previous and present studies clearly demonstrated that there may be different pathways contributing to extra-territorial tactile allodynia/hyperalgesia via astrocytes or microglia. Although the priority of these different pathways remains to be established, intrathecal injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by TNF-α, induced mechanical allodynia in the paw (Gao et al., 2010). Therefore, there is a possibility that sTNF-α released from microglia following P2X 7 activation may activate astrocytes and induce the release of IL-1β in this MNT model. "

[Show abstract][Hide abstract]ABSTRACT: The whisker pad area (WP) is innervated by the second branch of the trigeminal nerve and experiences allodynia and hyperalgesia following transection of the mental nerve (MN; the third branch of the trigeminal nerve). However, the mechanisms of this extra-territorial pain remain unclear. The ionotropic P2X(7) ATP receptor (P2X(7)) in microglia is known to potentiate, via cytokines, the perception of noxious stimuli, raising the possibility that P2X(7) and cytokines are involved in this extra-territorial pain. One day after MN transection (MNT), WP allodynia/hyperalgesia developed, which lasted for > 8 wks. Activation of microglia and up-regulation of P2X(7), membrane-bound tumor necrosis factor (TNF)-α (mTNF-α), and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC) were evident for up to 6 wks after MNT. Allodynia/hyperalgesia after MNT was blocked by intracisternal administration of etanercept, a recombinant TNF-α receptor (p75)-Fc fusion protein. Intracisternal A438079, a P2X(7) antagonist, also attenuated allodynia/hyperalgesia and blocked up-regulation of mTNF-α and sTNF-α in the TNC. We conclude that sTNF-α released by microglia following P2X(7) activation may be important in both the initiation and maintenance of extra-territorial pain after MNT.